【作者】 王刚；

【导师】 孙康； 窦红静；

【作者基本信息】 上海交通大学 ， 材料学， 2013， 博士

【摘要】 聚合物微球具有比表面积大、粒径形貌可控、表面可灵活修饰多种官能团等特点，是一种十分适合生物医学应用的载体。无机纳米颗粒作为纳米材料的重要组成部分，由于其特殊的尺寸效应而具有独特的功能性。因此，将无机纳米颗粒与聚合物微球相结合所形成的新型复合微球能够兼具两者的特性，从而在生物医学领域有着巨大的应用潜力。这其中，基于荧光编码微球的液相悬浮式生物芯片，作为一项新兴生物检测技术，因其高通量，多元化等特点而受到广泛关注。本文的工作主要围绕着基于无机/聚合物复合微球的液相悬浮式生物芯片体系的构建及应用进行相关研究，具体开展了以下工作。（1）将传统的分散聚合法、种子聚合法、乳液溶剂挥发法与新技术膜乳化法相结合，可控地制备了具有不同结构形貌的单分散表面羧基化聚合物微球。首先以分散聚合制备的单分散聚苯乙烯微球作为种子，利用种子聚合法制备出了单分散聚（苯乙烯-二乙烯基苯-甲基丙烯酸）多孔微球。微球的孔结构与粒径可由种子微球的性质和交联剂浓度来进行控制，且带有来自于甲基丙烯酸的羧基。之后又利用膜乳化-乳液溶剂挥发法制备了无孔的聚（苯乙烯-马来酸酐）共聚物微球。在合适的膜乳化条件下，微球单分散性良好且粒径大小可由膜孔径来进行控制，通过对微球进行表面酸催化水解改性则可使微球表面带有羧基官能团。最后又以膜乳化-乳液溶剂挥发法制备出的单分散聚苯乙烯微球作为种子，利用种子聚合法制备了单分散表面羧基化的聚（苯乙烯-二乙烯基苯-甲基丙烯酸）中空微球，并通过调节种子微球的分子量实现了对中空微球的形貌控制。（2）根据制备聚合物微球的方法机理以及所得微球的结构特点,我们采用不同方法进行无机纳米颗粒/聚合物复合微球的制备。首先利用所制备的单分散聚（苯乙烯-二乙烯基苯-甲基丙烯酸）微球的多孔结构，在以往溶胀法渗透包埋的原理基础上，设计了以微球内外高量子点浓度差为主要驱动力的溶胀挥发法来进行量子点复合荧光微球的制备。此方法相对传统溶胀法在对量子点的包埋和保护上具有更好效果，并可通过调节溶胀溶液中量子点的种类与浓度使微球具有不同的荧光发射光谱。在此基础上，以一步制备无机/聚合物复合微球为设计思想，我们又将膜乳化技术引入复合微球的制备。与溶胀挥发法相比，其所制备的量子点复合荧光微球具有更好的荧光时间稳定性，能够在不同温度和pH值环境中保持良好的荧光性能，并可通过简单地调节分散相中量子点的种类与浓度使微球具有多样的荧光发射光谱。最后，鉴于膜乳化-乳液溶剂挥发法的方法优势，我们还进一步通过在分散相中同时加入Fe3O4磁性纳米颗粒与量子点，制备了具有磁性和荧光双功能特性的复合微球。（3）基于自制的量子点复合荧光微球，我们初步构建了一个以流式细胞仪作为检测平台的液相悬浮式生物芯片检测体系。首先创新性地引入与微球尺寸相关的前向散射（FS）信号，结合微球在FL1和FL4两个波段荧光检测通道中的荧光信号，建立起了具有30个编码的三维微球编码库。之后，以乙肝病毒的五种抗原/抗体作为检测对象，利用流式细胞仪进行了实验方案的设计以及乙肝病毒的免疫检测。通过对乙肝病毒五种血清标记物所进行的单因子免疫检测，交叉反应检测以及多因子免疫检测实验，我们成功获得了多条具有良好特异性与灵敏度的检测信号-检测目标物浓度相关曲线。以此为基础，我们还对人血清样品进行了测试，并与商用临床诊断技术进行了比较。最终我们从结果上验证了自制液相悬浮式生物芯片体系在生物医学检测上的可行性和有效性。此外，我们还利用自制磁性荧光微球成功地对乙肝病毒HBsAg分子进行了特异性磁分离，从而进一步提高了自制复合微球在生物医学领域的潜在应用价值。在综上全文三大部分的研究中，我们在如下方面得到了新颖的研究结果：1）在量子点复合荧光微球的制备中，提出了一种溶胀-挥发法来提高所得微球的荧光性能，并通过该方法与传统溶胀法的比较证实了其在制备量子点复合荧光微球中的优势。2）将SPG膜乳化法用于无机纳米颗粒/聚合物复合微球的制备。该方法条件温和、制备效率高的特点使其在量子点复合荧光微球的制备中具有突出优势，所得微球尺度均一可控、荧光性能优异。3）在量子点复合微球编码库的建立中，基于流式细胞仪检测平台，利用膜乳化法在微球尺寸调节中的优势，将FS这一与尺寸相关的信号作为新型编码参数，从而丰富了编码库中微球编码的数目，最终成功设计并初步实现了量子点复合编码微球在乙肝病毒多因子免疫检测中的应用。更多还原

【Abstract】 Polymeric microspheres are well-suited carriers in biomedicalapplications for their large specific surface area, controllable size andcapability to be modified with various functional groups. In another hand,as one kind of important nano-materials, inorganic nanoparticles haveunique properties for their special size effect. Therefore, inorganicnanoparticles/polymeric composite microspheres can combine thesefunctional characteristics from both compositions, and possess greatpotential value in biomedical application fields. Especially, a biomedicalassay technology named suspension biochips, which was based onfluorescent microspheres, attracted much attention in recent years for itshigh-throughput and multiplex technical features. So in this work, wefocused on the establishment and application of inorganic/polymericcomposite microspheres based suspension biochip system, and thecontributions are as follows:(1) We achieved controllable fabrication of monodisperse carboxylcapped polymeric microspheres with various morphologies by integratingdispersion polymerization, seed polymerization, emulsion solvent evaporation approach and membrane emulsification technology. First,porous poly(St-DVB-MAA) microspheres were fabricated via seedpolymerization with monodisperse PS seeds synthesized by dispersionpolymerization. The porous structure and size of microspheres could beboth controlled by the properties of seeds and crosslinker concentrations.Meanwhile, the microspheres had carboxyl groups which come from theMAA added. Thereafter, we fabricated nonporous PSMA microsphereswith membrane emulsification-emulsion solvent evaporation method(MEESE method). Under proper emulsification conditions, the size ofPSMA microspheres was determined by the pore size of membrane, andthe surface anhydride groups of PSMA microspheres could be tranferredinto carboxyl groups via acid-catalyzed hydrolysis. Finally, hollowpoly(St-DVB-MAA) microspheres were synthesised via seedpolymerization with PS seeds fabricated in MEESE method. Themorphologies of these hollow microspheres could be controlled by themolecular weights of seeds.(2) According to the principle for fabricating polymeric microspheresand the structures of resultant polymeric microspheres, different methodswere used to fabricate inorganic/polymeric composite microspheres. First,a swelling-evaporation method (SE method) was developed to fabricateQD-microspheres with porous poly(St-DVB-MAA) microspheres, inwhich the swelling process was combined with gradual evaporation of thesolvent, thus gradual concentration of QDs in the dispersion solution. Thismethod was demonstrated to be an efficient method for improving thefluorescence intensity of resultant microspheres compared with the use ofswelling alone. Through this method, QD-microspheres encoded witheither single or multiple wavelength-emitting fluorescence spectra werefabricated effectively by adding different QDs into the swelling solution.Thereafter, to achieve a one-step fabrication, we developed an efficientMembrane Emulsification-Emulsion Solvent Evaporation (MEESE)method to fabricate the QD-microspheres. The QD-microspheres fabricated in this method showed excellent fluorescence stability in variousenvironments. And the fluorescence spectra of QD-microspheres could beconveniently controlled by changing the QDs in the dispersion phase.Finally, to further take the advantages of MEESE method, bi-functionalcomposite microspheres were successfully fabricated by adding magneticFe3O4nanoparticles and QDs into dispersion phase simultaneously.(3) Based on the self-fabricated QD-microspheres, we established asuspension biochip system on the flow cytometric platform. First, by thecombination of the advantages of MEESE approach in controllingQD-microsphere size and accurate encoding, a three dimensional barcodelibrary with the integration of signals in Forward Scatter (FS), FL1andFL4Channels was established in flow cytometer. Thereafter，the fiveindexes of hepatitis B viruses (HBV) were chosen as diagnosis targets，andthe detection experiments of HBV immunoassays were designed andcarried out in a flow cytometer. Based on the successful implementation ofsingleplex, cross-reaction and multiplex immunoassays of five HBV serummarkers, detection standard curves of HBV were established whichshowed good specificity and sensitivity. Then, in comparison withcommercial clinical detection technology, detections of human serumsamples were carried out. Finally, all the detection results verified that theself-established suspension biochip system was feasible and effective inthe biomedical assays. Beside these, by using self-fabricated magnetic andfluorescent microspheres, a specific magnetic separation for HBsAg wasachieved, which further promoted the applications of self-fabricatedcomposite microspheres in biomedical fields.In summary, the conclusions of innovations in our work are as follows:1) In the fabrication of QD-microspheres, a SE method was developed toimprove the fluorescence properties of QD-microspheres, and theadvantages of this method were further verified by comparison withconventional swelling method.2) The SPG membrane emulsificationtechnology was introduced into the fabrication of inorganic/polymeric composite microspheres. This method has unique advantages owing to itsmild fabrication conditions and high productivity. The resultant compositemicrospheres have good monodipersity, controllable size and excellentfluorescence properties.3) In the set-up of barcode library ofQD-microspheres, we take the advantage of membrane emulsification inthe size controlling of QD-microspheres, and the flow cytometric FS signal,which was correlative to the size of QD-microspheres, was introduced as anew encoding parameter for more barcodes. Finally, a QD-microspherebased suspension biochip system was firstly designed and applied to theimmunoassays of HBV successfully.更多还原